Method for inducing death of neoplastic cells using piperazne derivatives

ABSTRACT

A method for inducing cell death in neoplastic cells, includes the step of administering a compound of formula I or a pharmaceutically acceptable salt thereof, as a primary chemotherapeutic agent or substantially contemporaneously with chemotherapy, to a patient having multidrug-resistant neoplastic cells, in an amount sufficient to induce cell death in the neoplastic cells: ##STR1## where R 1  is hydroxyl, C1-4 alkoxyl, C1-4 alkylcarboxyloxymethoxyl, phenyl C1-2 alkylamino group, 2,5-pyrrolidinedione-1-alkoxyl (C1-4), or ##STR2## wherein X 1  is a chemical bond or C1-2 alkylene, X 2  is hydrogen or carboxyl forming a 5-membered ring with X 1  when X 2  is methylene, X 3  is hydrogen or C1-2 alkyl, X 4  is hydrogen or C1-2 alkyl, or X 3  and X 4  together form a 5-membered ring, in which at least one of X 2 , X 3 , and X 4  is hydrogen, 
     R 2  is C3-4 alkyl, R 3  is C1-4 alkyl, ##STR3##  in which n is an integer of 0 to 3.

BACKGROUND

1. Field of the Invention

This invention relates to a method for inducing death of neoplasticcells and potentiating chemotherapy to treat neoplastic cells, andparticularly to such a method for activating programmed cell death ofneoplastic cells and reversing multidrug resistance in neoplastic cells,using piperazine derivatives.

2. Background of the Art

Certain epoxide compounds are known as pharmacologically activecompounds in a variety of pharmaceutical fields, including in the fieldof cancer treatment. Numerous epoxide compounds have been synthesizedfor various purposes. For example, U.S. Pat. Nos. 4,507,297 and4,596,803 to Masaki et al. disclose piperazine derivatives having anepoxide group including NCO-700 used for the purpose of inhibitingmyocardial infarction. U.S. Pat. No. 5,336,783 to Omura et al. disclosesa pyrrole derivative having a carbamoyl group, which has calpaininhibitory activity. U.S. Pat. No. 4,732,910 to Yaginuma et al.discloses an epoxide compound having a guanidino group and a benzylgroup, which has strong enzyme inhibitory activity against thiolproteases. U.S. Pat. Nos. 4,333,879 and 4,382,889 to Tamai et al.disclose EST as a compound having thiol protease inhibitory activity,especially calcium-activated neutral protease (CANP) inhibitoryactivity. U.S. Pat. Nos. 4,418,075 and 4,474,800 to Tamai et al.disclose compounds having a similar chemical structure to that of EST,but which contains one more imino group than EST does.

The compounds described in the preceding paragraph have thiol proteaseinhibitory activity, especially against CANP (also known as calpain).However, none of the above compounds, have been reported as effective inthe direct treatment of cancer cells, despite the fact that calpaininhibitors have some pharmaceutical effects on humans.

A number of other calpain inhibitors have been disclosed for variousutilities. For Example, U.S. Pat. No. 5,403,834 to Malfroy-Camine et al.discloses salen-transition metal complexes which have potent antioxidantand/or free radical scavenging properties. The compounds are said toprevent or reduce ischemic/reperfusion damage to critical tissues suchas the myocardium and central nervous system. U.S. Pat. No. 5,328,922 toNixon, et al. discloses two related endogenous neural, especially humanbrain, calcium-activated neutral protease (CANP or calpain) inhibitorswhich are known as high molecular weight calpastatin (HMWC) and lowmolecular weight calpastatin (LMWC). U.S. Pat. No. 5,268,164 toKozarich, et al. discloses peptides (permeabilizer A-7) having a coresequence of amino acids or amino acid analogues which increase thepermeability of the blood-brain barrier in an animal. U.S. Pat. Nos.5,424,325, 5,422,359, 5,416,117, 5,395,958, and 5,340,909 to Ando et al.disclose aminoketone derivatives ('325), alpha-aminoketone derivatives('359), and cyclopropenone derivatives ('117, '958, and '909), which arereversible inhibitors against thiol proteases, such as calpain. Thesecompounds are said to have excellent properties in tissue distribution,cell membrane permeability, and absorption on oral administration.Treatment of cancer cells is not mentioned in connection with thecompounds.

International Application Publication No. WO 94/00095 discloses the useof various calpain inhibitors in order to synchronize the cell cycle.The synchronization is disclosed to shorten the duration of chemotherapyfor cancer and to increase the activity of chemotherapeutic agents. Therationale is that synchronization of all cells in the S phase willrender them more sensitive to the chemotherapeutic agents. Thisreference teaches that, in the use of calpain inhibitors in thetreatment of cancer, calpain inhibitors must be used prior to treatmentof cancer with a primary chemotherapeutic agent. Further, there is nodisclosure or suggestion of treatment of cancer cells which havemultidrug resistance. Moreover, in no way is there any disclosure orsuggestion of administering the calpain inhibitors by themselves toinduce cell death in cancer, i.e., the calpain inhibitors are notdisclosed to have a beneficial effect unless they are administered priorto treatment of cancer with a primary chemotherapeutic agent.

Shoji-Kasai et al. Proc. Natl. Acad. Sci., USA 85:146-150 (1988) showsthat epidermoid carcinoma A431 cells cultured in a chemically definedmedium can be arrested at mitotic metaphase by E-64-d which is amembrane-permeant derivative of the thiol protease-specific inhibitorE-64. This reference shows that inhibitors of CANP appear to havesignificant effects in slowing the growth of certain cancers. However,the effects will not be exhibited when cancer cells have multidrugresistance, and will not lead to cell death in cancer.

A major factor limiting the clinical usefulness of anticancer drugs isthe development of drug resistance in tumors. Many tumors which aretreated with anticancer drugs such as vinca alkaloids (vinblastine) andantracyclines (doxorubicin) develop tolerance to these drugs and alsoshow cross-resistance to other cancer drugs as well. In some cases,patients do not respond to initial chemotherapy at all, and in thesecases it is thought that the neoplasms have intrinsic drug-resistance.One of the mechanisms of this drug resistance is thought to reside inthe active pumping of cancer drugs out of the cancer cell by a surfaceprotein called the mdr (multiple drug resistance) protein (Gottesman etal., J Clinical Oncology 7:409-411, 1989, Goldstein et al., J Nat'lCancer Inst. 81: 116-124, 1989, Fojo et al., Cancer Res. 45:3002-3007,1985). This protein effectively lowers the concentration of anticancerdrugs within the cancer cell leading to survival and growth of thetumor. The mdr protein has been characterized as a 170,000 dalton,energy-dependent glycoprotein, which becomes highly amplified in cancercells as they acquire drug resistance. The pump is thought to recognizeand efflux a number of hydrophobic drugs and its normal role in the bodyis thought to be as a pump which can recognize and rid cells ofpotentially toxic compounds.

A number of chemical compounds have been reported to block the activityof the mdr pump allowing anticancer drugs to accumulate in the targetcells. These compounds are thought to work by acting as substrates forthe multidrug pump system, and by overwhelming the pump to prevent theefflux of cancer drugs resulting in cancer cell death. The mostintensely studied of these mdr-blocking compounds has been verapamil, acalcium-channel blocker which has a different clinical use, namely thetreatment of hypertension. In both preclinical and clinical studies(Gottesman et al., J Clinical Oncology 7:40-411, 1989), verapamil hasshown good activity in inhibiting the mdr pump, resulting in enhancedkilling of cancer cells. Unfortunately, the doses of verapamil needed toinhibit the pump are so high that toxic cardiovascular side effects wereseen in patients, thus preventing the further development of this drugfor use in cancer patients. Nevertheless, the development of nontoxicagents that can reverse the mdr pump are needed and would be animportant addition to the treatment of cancer patients. As shown inTable 1 below, the number of cancer patients in the United States whodevelop drug resistance is approximately 30% and, thus, the market forsafe, effective blockers of mdr pump is significant.

                  TABLE 1                                                         ______________________________________                                        Drug Resistance in U.S. Cancer Patients.sup.1                                 Number of Cancer Patients (U.S.)                                                                        1,200,000                                           Number of New Patients/Year                                                                             900,000                                             Estimated Number of Patients with Drug-Resistant                              Cancer                    350,000                                             ______________________________________                                         .sup.1 Numbers quoted from BioTechnology News and Journal of the National     Cancer Institute                                                         

With regard to treatment of multidrug resistant cancer cells, U.S. Pat.No. 5,371,081 discloses N-substituted phenoxazines. These compounds arechemically unrelated to the above-discussed epoxide compounds andcalpain inhibitors. Further, the compounds are not disclosed to bebeneficial when administered by themselves, and the toxicity of thecompounds are likely to be an obstacle to clinical use.

One of the most serious problems residing in conventional chemotherapyis the toxicity of chemotheraputic agents. For example, vinblastine andadriamycin, typifying chemotheraputic agents, have inevitable sideeffects such as hair loss, weight loss, and liver and kidney damage.They cannot be administered daily for a long period of time due to hightoxicity. For example, these chemotherapeutic agents are normallyadministered several days a week for two or three months, and afterseveral weeks of recuperation without administration of thechemotherapeutic agents, the administration thereof is repeated. None ofthe conventional chemotherapeutic agents and potentiators is free oftoxicity.

In conclusion, no prior art discloses or suggests calpain inhibitorycompounds or epoxide compounds which themselves are practically capableof inducing cell death in cancer. Moreover, there is no disclosure ofcompounds which function to kill cancer cells, irrespective of theexistence of multidrug resistance in the cancer cells, withoutsignificant side effects or toxicity.

SUMMARY OF THE INVENTION

The present invention has exploited use of epoxide derivatives in thetreatment of cancer, and in particular, in order to treat cancer cells,irrespective of the existence of multidrug resistance in the cancercells, use of piperazine derivatives having epoxy groups as a primarychemotherapeutic agent or as adjunctive therapy when used substantiallycontemporaneously with other chemotherapeutic agents. An objective ofthe present invention is to provide a method for significant inductionof cell death in cancer, using piperazine derivatives having epoxygroups, which is pharmacologically beyond prevention of metastasis incancer or the like mentioned in the prior art. Another objective of thepresent invention is to provide a method for significant induction ofcell death in cancer, using piperazine derivatives having epoxy groupsalone or as a primary chemotherapeutic agent. In other words, thepiperazine derivatives themselves function as an anticancer drug,irrespective of the existence of multidrug resistance in the cancercells, based on a mechanism different from that of conventionalanticancer drugs. Still another objective of the present invention is toprovide a method for reversal of multidrug resistance in cancer cells byusing piperazine derivatives having epoxy groups as adjunctive therapywhen used substantially contemporaneously with other chemotherapeuticagents.

Namely, one important aspect of the present invention is a method forinducing cell death in neoplastic cells, comprising: administering acompound of formula I or a pharmaceutically acceptable salt thereof, toa patient having neoplastic cells, in an amount sufficient to inducecell death in said neoplastic cells: ##STR4## where R¹ is hydroxyl, C1-4alkoxyl, C1-4 alkylcarbonyloxymethoxyl, phenyl C1-2 alkylamino group,2,5-pyrrolidinedione-1-alkoxyl (C1-4), or ##STR5## wherein X¹ is achemical bond or C1-2 alkylene, X² is hydrogen or carboxyl forming a5-membered ring with X¹ when X¹ is methylene, X³ is hydrogen or C1-2alkyl, X⁴ is hydrogen or C1-2 alkyl, or X³ and X⁴ together form a5-membered ring, in which at least one of X², X³, and X⁴ is hydrogen,

R² is C3-4 alkyl, R³ is C1-4 alkyl, ##STR6## in which n is an integer of0 to 3.

In the above method, the compound is preferably of the followingformula: ##STR7## where R⁴ is selected from the group consisting of:##STR8##

Also, in the above method, the compound is preferably of the followingformula: ##STR9## where R⁴ is selected from the group consisting of:##STR10##

The above compounds are effective in inducing cell death in cancer, evenwhen the cancer cells have multidrug resistance. The compounds arepreferably in the form of sulfate.

The above compounds include the piperazine derivatives disclosed in U.S.Pat. Nos. 4,507,297 and 4,596,803 to Masaki et al, and Japanese PatentLaid-open Nos. 63-275575 (1988) and 63-275576 (1988). These patents arehereby incorporated herein by reference. However, the piperazinederivatives in '297 and '803 have been reported only for the purpose ofinhibiting myocardial infarction. The above particular group ofpiperazine derivatives exhibits surprisingly and unexpectedly highanti-neoplastic activity on neoplasms, especially on those havingmultidrug resistance. The neoplasms which can be effectively treated areselected from the group consisting of human breast cancer cells, humanmelanoma cells, human ovarian cancer cells, human colon cancer cells,human pancreatic cancer cells, and human prostate cancer cells,particularly undifferentiated cancer cells.

Another important aspect of the present invention is a method fortreating neoplastic cells, consisting essentially of administering acomposition consisting essentially of a compound of formula I indicatedearlier or a pharmaceutically acceptable salt thereof to a patienthaving multidrug-resistant neoplastic cells, in an amount sufficient toinduce cell death in said neoplastic cells. The preferred compounds andthe effective neoplasms described earlier can be adopted in the abovemethod. The piperazine derivatives themselves in the present inventiondemonstrate significantly high anti-neoplastic activity on cancer cells,even when no other chemotherapeutic agents are used. This unexpectedfinding appears to be based on "apoptosis", i.e., programmed cell deathwhich only recently has been recognized (Desoize B., Anticancer Res.14:221-2294, 1994), in addition to the reversal of multidrug resistanceexpressed by an active mdr gene. The significant induction of cell deathin cancer, via its apoptosis and reversal of multidrug resistance, bythe piperazine derivatives in the present invention is pharmacologicallyvery different from and beyond, for example, the reported prevention ofmetastasis in cancer by thiol protease-specific inhibitor E-64-d whichhas an epoxy group but no piperazine ring (Shoji-Kasai et al. Proc.Natl. Acad. Sci., USA 85:146-150, 1988). Further, the use of thepiperazine derivatives in the present invention as a primarychemotherapeutic agent or as adjunctive therapy when used substantiallycontemporaneously with other chemotherapeutic agents is very distinctfrom the reported use of calpain inhibitors which must be used prior totreatment for cancer with a primary chemotherapeutic agent(International Application Publication No. WO 94/00095).

The above piperazine derivatives are also effective in conjunction withthe use of a chemotherapeutic agent such as vinblastine and adriamycinby administering the piperazine derivatives substantiallycontemporaneously with the chemotherapeutic agent to a patient havingneoplastic cells, particularly those carrying an active mdr gene.

Thus, another important aspect of the present invention is a method forpotentiating chemotherapy to treat neoplastic cells, comprisingadministering a compound of formula I indicated earlier or apharmaceutically acceptable salt thereof to a patient havingmultidrug-resistant neoplastic cells, substantially contemporaneouslywith chemotherapy, in an amount sufficient to potentiate saidchemotherapy. The preferred compounds and the effective neoplasmsdescribed earlier can be adopted in the above method. The piperazinederivatives in the present invention are highly effective in reversingdrug resistance in human cancer cells and tumors that are resistant toanticancer drugs, via highly accumulating the anticancer drugs in thesecancer cells, thereby increasing the killing of these cancer cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the relationship between LDH released and theconcentration of NCO-700 in cancer cell death assays of human ovariancancer cell lines SW-626 and AN-3-CA.

FIG. 2 is a graph showing the relationship between LDH released and theconcentration of NCO-700 in cancer cell death assays of melanoma celllines B16F10 (mouse line) and SK-Mel-2 (human line).

FIG. 3 is a graph showing the relationship between LDH released and theconcentration of NCO-700 in cancer cell death assays of human cell lineSK-Mel-2 (melanoma) of 100,000 cells and 250,000 cells.

FIG. 4 is a graph showing the relationship between LDH released and theconcentration of NCO-700 in cancer cell death assays of human cancercell lines MCF-7 (breast), HL-60 (leukemia), and HEP-129 (liver).

FIG. 5 is a graph showing the relationship between LDH released and theconcentration of NCO-700 in cancer cell death assays of human cancercell lines HS-578T (breast), T-47D (breast), and DU-145 (prostate).

FIG. 6 is a graph showing the relationship between LDH released and theconcentration of NCO-700 in cancer cell death assays of human cancercell lines MCF-7 (breast), MDA-MB231 (breast), and LS-174T (colon).

FIG. 7 is a graph showing the relationship between LDH released and theconcentration of NCO-700, TOP-008, TOP-009, TOP-013, and TOP-017 incancer cell death assays of human cancer cell line HS-578T (breast).

FIG. 8 is a graph showing the relationship between LDH released and theconcentration of TOP-008 in cancer cell death assays of human cancercell lines HS-578T (breast), DU-145 (prostate), PC-3 (prostate), andWIDR (colon).

FIG. 9 is a graph showing the relationship between the object area (thenumber of surviving cancer cells calculated by a computer) and theconcentration of NCO-700 in a cancer cell survival assay of human cancercell line SK-Mel-2 (melanoma).

FIG. 10 is a graph showing the relationship between the object area andthe concentration of NCO-700 in a cancer cell survival assay of humancancer cell line LS-174T (colon).

FIG. 11 is a graph showing the relationship between the object area andthe concentration of NCO-700 in a cancer cell survival assay of humancancer cell line T-47D (breast).

FIG. 12 is a graph showing the relationship between the object area andthe concentration of NCO-700 in a cancer cell survival assay of humancancer cell line HS-578T (breast).

FIG. 13 is a graph showing the relationship between the object area andthe concentration of NCO-700 in cancer cell survival assays of humancancer cell line DU-145 (prostate) of 500 cells/well and 1,000cells/well.

FIG. 14 is a graph showing the relationship between the object area andthe concentration of NCO-700 in cancer cell survival assays of humancancer cell line PC-3 (prostate) of 500 cells/well and 1,000 cells/well.

FIG. 15 is a graph showing the relationship between the object area andthe concentration of NCO-700 in cancer cell survival assays of humancancer cell line LN (prostate).

FIG. 16 is a graph showing the relationship between the object area andthe concentration of NCO-700 in cancer cell survival assays of humancancer cell line WIDR (colon) of 1,000 cells/well.

FIG. 17 is a graph showing the relationship between the mean change insize of tumor (delta mean) and the dosage of NCO-700 in a sub-renalcapsule assay of human prostate cancer in BDF/1 mice.

FIG. 18 is a graph showing the relationship between the change in sizeof tumor and the dosage of NCO-700 in a sub-renal capsule assay of humanbreast cancer in BDF/1 mice.

FIG. 19 is a graph showing the relationship between the change in sizeof tumor and the dosage of NCO-700 in a sub-renal capsule assay of humancolon cancer in BDF/1 mice.

FIG. 20 is a graph showing the LDH release stimulated by NCO-700,TOP-008, and analogs in cancer cell death assays of human cancer cellline DU-145 (prostate) at a concentration of 25 μM.

FIG. 21 is a graph showing the LDH release stimulated by NCO-700,TOP-008, and analogs in cancer cell death assays of human cancer cellline HS-578T (breast) at a concentration of 25 μM.

FIG. 22 is a graph showing the LDH release stimulated by NCO-700,TOP-008, and analogs in cancer cell death assays of human cancer cellline T-47D (breast) at a concentration of 25 μM.

FIG. 23 is a graph showing the LDH release stimulated by NCO-700,TOP-008, and analogs in cancer cell death assays of human cancer cellline SK-MEL-2 (melanoma) at a concentration of 25 μM.

FIG. 24 is a graph showing the LDH release stimulated by NCO-700,TOP-008, and analogs in cancer cell death assays of human cancer cellline WIDR at a concentration of 25 μM.

FIG. 25 is a graph showing the LDH release stimulated by NCO-700,TOP-008, and analogs in cancer cell death assays of human cancer cellline LS-174T (colon) at a concentration of 25 μM.

FIG. 26 is a graph showing the relationship between the object area andthe concentration of TOP-008 in cancer cell survival assays of humancancer cell line HS-578T (breast) of 500 cells/well and 1,000cells/well.

FIG. 27 is a graph showing the relationship between the object area andthe concentration of TOP-008 in cancer cell survival assays of humancancer cell line T-47D (breast) of 500 cells/well and 1,000 cells/well.

FIG. 28 is a graph showing the relationship between the object area andthe concentration of TOP-008 in cancer cell survival assays of humancancer cell line DU-145 (prostate) of 500 cells/well and 1,000cells/well.

FIG. 29 is a graph showing the relationship between the object area andthe concentration of TOP-008 in cancer cell survival assays of humancancer cell line PC-3 (prostate) of 500 cells/well and 1,000 cells/well.

FIG. 30 is a graph showing the relationship between the object area andthe concentration of TOP-008 in cancer cell survival assays of humancancer cell line LS-174T (colon) of 1,000 cells/well and 2,000cells/well.

FIG. 31 is a graph showing the relationship between the object area andthe concentration of TOP-008 in cancer cell survival assays of humancancer cell line WIDR (colon) of 500 cells/well.

FIG. 32 is a graph showing the LDH release stimulated by NCO-700,TOP-008, and analogs in cancer cell death assays of human cancer celllines HS-766T (pancreas) and ASPC-1 (pancreas) at a concentration of 25μM.

FIG. 33 is a graph showing the relationship between LDH released and theconcentration of NCO-700 and Calpain inhibitor 1 in cancer cell deathassays of human cancer cell line DU-145 (prostate).

FIG. 34 is a graph showing the LDH release stimulated by NCO-700,TOP-008, and analogs in cancer cell death assays of human cancer cellline HS-578T (1,000 cells/well).

FIG. 35 is a graph showing the LDH release stimulated by NCO-700,TOP-008, and analogs in cancer cell death assays of human cancer cellline HS-578T (1,500 cells/well).

FIG. 36 is graphs showing the results of apoptosis activity of TOP-008in apoptosis detection assays of human cancer cell line HS-578T. FIGS.36a, 36b, and 36c indicate normal cancer cells, cancer cells withTOP-008 (50 μM), and cancer cells with TOP-008 (25 μM), respectively.

FIG. 37 is a graph showing the relationship between changes in tumorweight and the dosage of NCO-700 in nude mice treated with adriamycinand without adriamycin.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

We have discovered that piperazine derivatives having epoxy groups inthe present invention can be used to kill tumor cells in vivo. Ourstudies show that, when the derivatives are administered to nude micecarrying a drug-resistant human tumor, there is a decline, by 60%, forexample, of tumor mass without any corresponding loss of body weight.Moreover, the drugs are effective, either alone or in combinationtherapy, even against cancer cells exhibiting multi drug resistance.

As discussed hereinabove in the background of the invention, a majorfactor limiting the clinical usefulness of anticancer drugs is thedevelopment of drug resistance in tumors. Recent research indicates thattwo major mechanisms which underlie drug resistance in cancer cellsinclude (i) amplification of the mdr multigene family and (ii)inhibition of programmed cell death (apoptosis). The mechanism of drugresistance in cells where the mdr gene is amplified resides in theactive pumping of cancer drugs out of the cancer cell by the mdr proteinwhich effectively lowers the concentration of anticancer drugs withinthe cancer cell. The second major mechanism of drug resistance in cancercells, which only recently has been recognized, is the inhibition ofprogrammed cell death, or apoptosis, which leads to resistance not onlyto chemotherapy, but radiation therapy as well (Desoize B., AnticancerRes. 14:221-2294, 1994). The best-characterized inhibitor of apoptosisis the protein product of the Bcl-2 gene, which is over-expressed incancer cells that are drug and radiation resistant. A variety of rapidand distinct molecular events occur in apoptotic cells, such as nuclearchromatin condensation, changes in cell size, and activation ofendogenous endonuclease activity that results in the production ofoligosomal DNA fragments. Multidrug-resistant cancer cells have theapoptosis inhibition activity.

We have discovered that the piperazine derivatives having epoxy groupsin the present invention are highly effective in reversing drugresistance in human cancer cells and tumors that are resistant toanticancer drugs. The derivatives can stimulate 5-fold the accumulationof the anticancer drug, vinblastine, in human carcinoma cell lines, forexample. This results in a highly significant increase in the killing ofthese cancer cells as determined in a cancer cell survival assay. Thesestudies indicate the piperazine derivatives having epoxy groups havesignificant activity in sensitizing drug-resistant cancer cells to thekilling effect of anticancer drugs and demonstrates the high potentialthereof for use in cancer patients.

As discussed in greater detail below, it is believed that thepharmaceutical compositions of the present invention are effective ininducing apoptosis in cancer cells. Thus, these compositions areadvantageously effective in overcoming both of the major knownmechanisms of drug resistance in cancer patients.

Piperazine Derivatives Having Epoxy Groups

The piperazine derivatives having epoxy groups in the present inventionare compounds represented by formula I, ##STR11## where R¹ is hydroxyl,C1-4 alkoxyl, C1-4 alkylcarbonyloxymethoxyl, phenyl C1-2 alkylaminogroup, 2,5-pyrrolidinedione-1-alkoxyl (C1-4), or ##STR12## wherein X¹ isa chemical bond or C1-2 alkylene, X² is hydrogen or carboxyl forming a5-membered ring with X¹ when X¹ is methylene, X³ is hydrogen or C1-2alkyl, X⁴ is hydrogen or C1-2 alkyl, or X³ and X⁴ together form a5-membered ring, in which at least one of X², X³, and X⁴ is hydrogen,

R² is C3-4 alkyl, R³ is C1-4 alkyl, ##STR13## in which n is an integerof 0 to 3.

The compounds represented by formula I are typically of the followingformula: ##STR14## where R⁴ is selected from the group consisting of:##STR15##

Also, other typical compounds are of the following formula: ##STR16##where R⁴ is selected from the group consisting of: ##STR17##

Examples of the compounds typically include the following substituents:

General Formula ##STR18## R¹ ##STR19## R² A: --(CH₂)₂ CH₃

B: --CH₂ CH(CH₃)₂

C: --C(CH₃)₃

R³ ##STR20##

Typical compounds included in the piperazine derivatives having epoxygroups are as follows:

    ______________________________________                                        No.    R.sup.1   R.sup.2                                                                             R.sup.3 Remarks                                        ______________________________________                                         1     A         B     A       trans, sodium                                   2     A         B     B       trans, sodium                                   3     A         B     C       trans, sodium                                   4     A         B     D       trans, sodium                                   5     A         B     D       (2R,3R), sodium                                 6     A         B     D       (2S,3S), sodium                                 7     A         B     E       trans, sodium                                   8     A         B     F       trans, sodium                                   9     A         B     G       trans, sodium                                  10     A         B     H       trans, sodium                                  11     A         B     H       trans, sodium                                  12     A         B     H       trans, sodium                                  13     B         B     D       trans, sodium                                  14     B         B     D       trans, sodium                                  15     B         B     D       (2R,3R), 1/2 sulfate                           16     B         B     F       (2R,3R), 1/2 sulfate                           17     B         A     D       (2R,3R), 1/2 sulfate                           18     B         A     F       (2R,3R), 1/2 sulfate                           19     B         C     D       (2R,3R), 1/2 sulfate                           20     B         C     F       (2R,3R), 1/2 sulfate                           21     C         B     D       trans, 1/2 sulfate                             22     D         B     D       (2R,3R), 1/2 sulfate                           23     E         B     D       (2R,3R), 1/2 sulfate                           24     F         B     D       (2R,3R), 1/2 sulfate                           25     G         B     D       (2R,3R), 1/2 sulfate                           26     H         B     D       (2R,3R), 1/2 sulfate                           27     H         B     F       (2R,3R), 1/2 sulfate                           28     I         B     F       (2R,3R), 1/2 sulfate                           29     J         B     F       (2R,3R), 1/2 sulfate                           30     K         B     F       (2R,3R), 1/2 sulfate                           31     L         B     D       (2R,3R), 1/2 sulfate                           32     L         B     F       (2R,3R), 1/2 sulfate                           33     M         B     D       (2R,3R), 1/2 sulfate                           34     N         B     D       (2R,3R), 1/2 sulfate                           35     O         B     D       (2R,3R), 1/2 sulfate                           36     O         B     F       (2R,3R), 1/2 sulfate                           37     P         B     D       (2R,3R), 1/2 sulfate                           38     Q         B     D       (2R,3R), 1/2 sulfate                           39     R         B     F       (2R,3R), 1/2 sulfate                           40     S         B     F       (2R,3R), 1/2 sulfate                           41     T         B     F       (2R,3R), 1/2 sulfate                           42     U         B     F       (2R,3R), 1/2 sulfate                           43     V         B     D       (2R,3R), 1/2 sulfate                           ______________________________________                                    

The specifically defined present piperazine derivatives having epoxygroups are believed to work through an epoxide group, but E-64 alsocontains an epoxide group. The Shoji-Kasai et al. reference describedearlier discloses that E-64-d analogous to E-64 exhibits relevanteffects, i.e., slowing the growth of cancer cells, and has a partiallysimilar structure to the exemplified compounds of the present invention.However, neither E-64 nor E-64-d contains a piperazine ring, and E-64has been found not to be particularly effective in cancer cells havingmultidrug resistance, despite the fact that it has been reported thatE-64-d slows the growth of cancer cells. It is believed that not onlyepoxy group but also a piperazine ring are essential to induction ofcell death in cancer. Calpain inhibitor I has been found slightlyeffective within the above context, but its toxicity may be an obstacle.Other compounds having similar structures to the present piperazinederivatives, with respect to piperazine rings when present, and epoxygroups, have been tested (data omitted here), but no significant effectwas observed, meaning that surprising and unexpected effects oninduction of cell death in cancer, i.e., reversing mdr and in inducingdeath of cancer cells without significant toxicity to normal tissues,which appear to be pharmacologically beyond prevention of metastasis incancer, are found in the specifically defined present compounds.

Production of Piperazine Derivatives

The piperazine derivatives represented in formula I in the presentinvention can be synthesized based on a usual acid halide method or amixed anhydride method, the details of which are set forth in U.S. Pat.Nos. 4,507,297 and 4,596,803 to Masaki et al., both entitled "PiperazineDerivatives and A Medicine Containing the Same", and Japanese PatentLaid-open Nos. 63-275575 (1988) and 63-275576 (1988), which are herebyincorporated herein by reference.

For example, in the case where R¹ in formula I is an alkoxyl group, aleucine derivative represented by the general formula (2), ##STR21##where R² is the same as in formula I, and R⁵ is a protective group foran amino group of an amino acid such as a tert-butoxycarbonyl group, orits reactive derivative, is reacted with an amino derivative representedby the general formula (3), ##STR22## where R³ is the same as definedabove, to obtain a compound represented by the general formula (4),##STR23## Subsequently, the protective group is removed by anyconventional method, and a leucylpiperazine derivative thus obtained andrepresented by the general formula (5), ##STR24## is reacted with atrans-epoxy succinic acid monoester represented by the general formula(6), ##STR25## where R¹ is the same as in formula I, or its reactivederivative, thereby obtaining a compound represented by the generalformula (7): ##STR26##

Alternatively, the trans-epoxy succinic acid monoester of the formula(6) above, or its reactive derivative, is reacted with leucine to obtainan epoxy succinyl leucine derivative represented by the general formula(8), ##STR27## where R¹ is the same as in formula I except that R¹ isnot hydroxyl group, or its reactive derivative. The compound of theformula (8) is then reacted with an amine derivative represented by theformula (3) above, thereby obtaining a compound of the formula (7)above.

In addition, the compounds of formula I can be obtained by the followingcondensation (dehydration) reaction: ##STR28##

The condensation reaction of the compound of the formula (2) with thecompound of the formula (3), the condensation reaction of the formula(5) with the compound of the formula (6) and the condensation reactionof the compound of the formula (8) with the compound of the formula (3)are conducted by a conventional acid halide method or a mixed anhydridemethod, or in an organic solvent such as methylene chloride, ethylenechloride, chloroform, ethyl acetate, tetrahydrofuran or the like in thepresence of a known condensation agent such as N-hydroxy succinimide andN,N'-dicyclohexylcarbodimide at -10° to +40° C., preferably at -5° to+30° C.

The ester residue of the compound represented by the formula (7) can bereadily converted to the corresponding carboxylic acid by any existingalkaline hydrolysis method.

A compound in which R¹ is a hydroxyl group can be obtained byhydrolyzing the ester group of the compound of the formula (7).

The piperazine derivative thus prepared may be optionally converted to apharmaceutically acceptable salt thereof, for example, of sodium,potassium, calcium or magnesium, or trialkylamine, dibenzlamine, N-loweralkylpiperidine, N-benzl-β-phenetylamine, α-phenethylamine,1-(1-naphthyl)ethylamine as well as hydrochloric acid, hydrobromic acid,formic acid, sulfuric acid, fumaric acid, maleic acid or tartaric acid.Further, with use of an optically active trans-epoxy succinic acidmonoester (6) such as a (2S,3S)-epoxy succinic acid monoester or a(2R,3R)-epoxysuccinic acid monoester which may be synthesized inaccordance with the method of Kenji Mori et al (Tetrahedron, vol. 36(1),87-90, 1980) or Japanese Patent Publication No. 3-18629 (1991), it ispossible to obtain the compounds of formula I of the present invention,which has an optically active epoxy succinic acid group, by the processnoted above.

Pharmaceutical Use

According to a further aspect of the invention, medicines are providedmedicines for induction of cell death in cancer, which medicines containthe compounds of formula I or their pharmaceutically acceptable salts asactive ingredients.

The usefulness of the compounds of formula I and their pharmaceuticallyacceptable salts according to the present invention as medicines forinduction of cell death in cancer has been confirmed by the fact thatthey have superior effects in inducing apoptosis and reversal ofmultidrug resistance of cancer cells.

Moreover, from acute toxicity tests using mice and rats, the compoundsof the invention were found to be quite safe for human use. For example,the LD₅₀ of NCO-700 via i.v. to rats is 317 mg/kg. This non-toxicity ofthe compounds makes it possible to administer the compounds to patientsdaily for a long period of time without interruption, withoutsignificant side effects, until the cancer tumors are suppressed. Thenon-toxicity, the apoptotic effects and the reversal of multidrugresistance of the compounds will change the procedures of conventionalchemotherapy completely.

The dosage of the compounds of formula I and their pharmaceuticallyacceptable salts varies depending upon the stage of cancer development,the type of cancer, the degree of multidrug resistance thereof, and thetype of chemotherapy which may be conducted concurrently. Generally,they may be administered to patients in an amount of from about 15 μg/kgto about 250 mg/kg, preferably from about 250 μg/kg to about 100 mg/kg,more preferably from about 1 mg/kg to about 50 mg/kg, to effectivelycause apoptosis and reverse multidrug resistance of cancer cells.Suitable target cancers are human breast cancer cells, human melanomacells, human ovarian cancer cells, human colon cancer cells, humanpancreatic cancer cells, human prostate cancer cells, especially whenthese cancer cells are in the undifferentiated stage.

For various formulations as medicines, the compounds of formula I andtheir salts may usually be combined with pharmaceutical carriers toprepare pharmaceutical compositions. Examples of the carriers includediluents or vehicles such as a filler, a binding agent, a disintegratorand a lubricant.

Such medicines are available in the dosage form of an injection, apowder, a capsule, a granule, a tablet or an ampule.

In the case of a tablet, a carrier is used which may be selected, forexample, from a vehicle such as lactose, saccharose, sodium chloride, aglucose solution, starch, calcium carbonate, crystal cellulose orsilicic acid; a binder such as water, ethanol, propanol, glucose, astarch solution, a gelatin solution, caraboxylmethyl cellulose, methylcellulose or potassium phosphate; a disintegrator such as dried starch,sodium alginate, an agar powder, sodium hydrogencarbonate, calciumcarbonate, stearic acid monoglyceride, starch or lactose; or a lubricantsuch as a stearate, a boric acid powder or solid polyethylene glycolwhich is known in the art. Where it becomes desirable, the tablet may besugar- or gelatin-coated, or film-coated.

In the case of an injection, a diluent is used which may be selected,for example, from water, ethyl alcohol, propylene glycol,polyoxyethylene sorbit or a sorbitan ester. In such instance, sodiumchloride, glucose or glycerine may be added in an amount sufficient toform an isotonic solution. A commonly used dissolving aid, a buffer, apain reliever or a preserving agent may also be convenientlyincorporated.

The present piperazine derivatives can be administered singly or incombination with a chemotherapeutic agent, or with other chemotherapysuch as radiation treatment. In the case of a combination of thepiperazine derivatives and a chemotherapeutic agent such as vinblastineand adriamycin, the piperazine derivatives can be administeredsubstantially contemporaneously with such a chemotherapeutic agent, sothat the piperazine derivatives can be formed into any pharmaceuticalforms uniformly with the chemotherapeutic agent.

This invention will be described in more detail with reference tocertain specific examples and test examples which are provided forpurposes of illustration only and are not construed as limiting. Eachtested compound was synthesized based on the production processexplained earlier. The test examples are intended to show the compoundsof formula I and their pharmaceutically acceptable salts as exhibitingsuperior effects on apoptosis and reversal of multidrug resistance ofcancer cells.

EXPERIMENT 1 Anti-neoplastic Activity of NCO-700 and Related AnalogsAlone ##STR29## Methods

In this experiment, three different assays were utilized to measureanti-neoplastic activity of compounds.

1. Cancer Cell Death Assay: Cell death was measured quantitatively bythe release of a marker intracellular enzyme, lactate dehydrogenase(LDH), from damaged or killed cells. LDH released into the extracellularfluid, was measured 24 hours after drug exposure to cancer cells and theenzyme activity was determined by the method of Decker et al. (J.Immunolog Methods 15:61-99, 1988).

2. Cell Survival Assays: Cell survival was assayed in cultured tumorcells. From 500-1000 cells per well were plated in 24-welled culturedishes and exposed to drug for seven days. After drug exposure, acomputerized cell imaging technique was utilized to ascertain the numberof viable cells remaining in each culture. From the data, survivalcurves were plotted and a ED₅₀ (drug concentration that permits 50% cellsurvival) were calculated.

3. In Vivo Model/Subrenal Capsule (SRC) Tumor Implant Assay: A tumorimplant model, where human tumors were implanted into the renal capsuleof mice was performed. The tumors successfully escaped the immune systemof the mouse and grew over a six-day period. The advantage of this assayis that the effect of chemotherapeutic agents can be tested in vivo.This model was developed at the University of California, Irvine byStratton et al. (Gynecologic Oncology 17:185-188, 1984). Human tumorswere implanted on day one and were exposed to drug on days two-six. Thesize of the implant was determined at time zero and at the end of fivedays of drug treatment, and the difference in size of the implantedtumor at day six, when compared to control animals not exposed to drug,is referred to herein as the delta mean.

Cell Lines Utilized

All cell lines used in the present study were obtained from the AmericanType Tissue Culture Laboratory and cultured according to theirspecifications. All were human cancer lines unless otherwise noted. Thefollowing cell lines and their tissue of origin used in our studieswere:

    ______________________________________                                        Cell Line        Tissue     Comment                                           ______________________________________                                        1.      MCF-7        breast     + estrogen rec                                2.      MDA-MB231    breast     + estrogen rec                                3.      T-47D        breast     + estrogen rec                                4.      HS-578T      breast     - estrogen rec                                5.      B16F10       melanoma   mouse line                                    6.      SK-Mel-2     melanoma                                                 7.      SW-626       ovarian                                                  8.      AN-3-CA      ovarian                                                  9.      HL-60        leukemia                                                 10.     Hep-129      liver                                                    11.     LS-174T      colon                                                    12.     WIDR         colon                                                    13.     DU-145       prostate   - testosterone rec                            14.     PC-3         prostate   - testosterone rec                            15.     LN           prostate   + testosterone rec                            ______________________________________                                    

Results

1) NCO-700 and Cancer Cell Death: The first assays performed measuredthe anti-neoplastic activity of NCO-700, alone, in cell death assays ofa wide variety of human cancer cells. The results of these experimentsare shown in FIGS. 1-6, where a dose-response curve of NCO-700 versus %LDH release is plotted, with higher values of LDH released indicatinggreater anti-neoplastic activity (apoptosis). From these assays, it canbe seen that (i) in breast cancer cells lines, the cells that wereresponding to NCO-700 were estrogen receptor negative, a trait generallyassociated with a more undifferentiated, and hence, more malignanttumor; (ii) in the melanoma group, all cells lines showed sensitivity toNCO-700; (iii) in the prostate cancer cell lines, the cell lines thatwere testosterone receptor negative (and more malignant) responded verywell to NCO-700; (iv) the colon and ovarian cancer cell lines werepartly responsive; and (v) the leukemia and liver cancer cell lines didnot respond to NCO-700. However, as shown in the experiment describedlater (Experiment 7), NCO-700 has significant anti-neoplastic activitieswhen used in conjunction with a standard chemotherapeutic agent such asvinblastine and adriamycin on drug-resistant tumors, even when NCO-700is not significantly effective in apoptosis of the tumors.

2) NCO-700 Analogs and Cell Death: A series of analogs related toNCO-700 were assayed in the cancer cell death assay. As shown in FIG. 7,a number of these analogs had significant anti-neoplastic activity asmeasured by LDH release from HS-578T, a breast cancer cell line. Whencompared to NCO-700, three analogs showed higher potency includinganalogs TOP-008, TOP-009 and TOP-013. In particular, TOP-008 showed thehighest potency with an approximately three-fold higher kill rate ofbreast cancer cells than NCO-700. It should be stressed that theseanalogs were tested alone, without any added chemotherapeutic agent.

As TOP-008 appeared to be the most potent NCO-700 analog tested to datein vitro, its anti-neoplastic activity was examined against other cancercell lines. In FIG. 8, a dose-response curve is shown of the effect ofTOP-008 on the killing of breast, prostate and colon cell lines. Thisanalog, on its own, shows excellent activity against these cancer celllines.

3) NCO-700 and Cancer Cell Survival Assay: The cancer cell survivalassay is an important additional in vitro assay to assess theanti-neoplastic activity of compounds as the cells are given a prolongedexposure (seven days) to the drug. FIGS. 9-16 show the effect ofincreasing the dose of NCO-700 on the survival of a number of cancercell lines. The survival studies confirm the results that were obtainedwith the cell death assay, namely that NCO-700 has significantanti-neoplastic activity, alone, against a number of human cancers(breast cancer cell lines T-47D and HS-578T, colon cancer cell linesLS-174T and WIDR, and prostate cancer cell lines DU-145, PC-3, and LN).In the figures, the vertical axis is the object area, which is thenumber of surviving cancer cells calculated by a computer.

4) NCO-700 Effect on Human Prostate Cancer Cells Grown In Vivo in theSRC Assay: Human cancer cells can be grown in the sub-renal capsule ofmice, where for a limited period of time they escaped the detection ofthe mouse's immune system. This assay provides a convenient system tostudy the effect of drugs on the growth of tumor cells in vivo. As thisis a much more time-consuming assay, and requires more drug than the invitro assays, the effect of NCO-700 was tested on a single prostatecancer cell line. Three groups of animals (n=23 total) were tested withthree concentrations of NCO-700 including, 10 mg/kg, 20 mg/kg and 40mg/kg. As shown in FIG. 17, there is a does response effect of NCO-700in decreasing the size of the tumor in the mice. Whereas the 10 mg/kghad no effect, there was a significant anti-neoplastic effect of NCO-700at the 20 mg/kg and 40 mg/kg level. At 40 mg/kg the effect of NCO-700was highly significant with a P value >0.001 (see FIG. 17). Again, theanti-tumor effect was seen with NCO-700 alone without any additionaldrugs added and points to the high potential of this and relatedcompounds as new anti-neoplastic agents.

5) NCO-700 Effect on Human Breast Cancer Cells Grown In Vivo in the SRCAssay: The effect of NCO-700 on a breast cancer line was tested in theSRC assay. Three groups of animals (n=18 total) were tested with threeconcentrations of NCO-700 including, 10 mg/kg, 25 mg/kg and 50 mg/kg. Asshown in FIG. 18, there is a dose response effect of NCO-700 indecreasing the size of the tumor in the mice. Whereas the 10 mg/kg hadno effect, there was a significant anti-neoplastic effect of NCO-700 atthe 25 mg/kg and 50 mg/kg level. These studies confirm the highpotential of NCO-700 and related compounds as new anti-neoplasticagents.

6) NCO-700 Effect on Human Colon Cancer Cells Grown In Vivo in the SRCAssay: The effect of NCO-700 on a colon cancer line was tested in theSRC assay. Three groups of animals (n=18 total) were tested with twoconcentrations of NCO-700 including 50 mg/kg and 100 mg/kg. As shown inFIG. 19, there is a dose response effect of NCO-700 in decreasing thesize of the tumor in the mice. Whereas the 50 mg/kg had slight effect,there was a significant anti-neoplastic effect of NCO-700 at the 100mg/kg level. These studies confirm the high potential of NCO-700 andrelated compounds as new anti-neoplastic agents.

Conclusion

Experiment 1 establishes that NCO-700, alone, is highly effective as ananti-neoplastic agent versus selective tumors. The NCO-700 analog,TOP-008, was three-fold more potent than NCO-700 in terms ofanti-neoplastic potency. The mechanism of NCO-700's anti-neoplasticactivity appears to be based on an apoptosis-related mechanism.

EXPERIMENT 2 Anti-neoplastic Activity of TOP-008 and Other NCO-700Analogs Alone ##STR30## Methods

As described in Experiment I, three different assays were performed tomeasure anti-neoplastic activity of compounds.

Results

1) NCO-700 Analogs and Cancer Cell Death: The first assays performedmeasured the anti-neoplastic activity of NCO-700 analogs in cell deathassays against a number of human cancer cells. The results of theseexperiments are shown in FIGS. 20-25, where a bar graph indicates theactivity of a particular analog as measured by the % LDH released, withhigher values of LDH released indicating greater anti-neoplasticactivity (apoptosis). All analogs were tested at a concentration of 25μM. From these assays, it can be seen that analogs of NCO-700 are highlyselective in their anti-neoplastic activity, with some analogs showingsignificantly greater activity than NCO-700 itself. The analogs willhave significant anti-neoplastic activities when used in conjunctionwith standard chemotherapeutic agents on drug-resistant tumors, evenwhen the analogs are not significantly effective in apoptosis of thetumors.

2) TOP-008 and Cancer Cell Survival Assay: FIGS. 26-31 show the effectof increasing the dose of TOP-008 against a number of cancer cell lines.The survival studies confirm that TOP-008 is a highly activeanti-neoplastic agent (apoptosis agent) and in some cases is much moreactive than NCO-700. For example, if the activity of NCO-700 and TOP-008are compared against the human colon cancer cell line WIDR (FIG. 16 inExperiment 1 versus FIG. 31) it is seen that the effective dose ofTOP-008 is much lower in this particular assay.

EXPERIMENT 3 Anti-neoplastic Activity of NCO-700 and Related AnalogsAlone on Pancreatic Cancer Cells

Methods

As described in Experiment 1, the cancer cell death assays wereperformed to measure anti-neoplastic activity of the compounds, NCO-700,TOP-001, TOP-003, TOP-005, TOP-006, TOP-008, TOP-009, TOP-010, TOP-012,TOP-013, TOP-015, and TOP-017.

Cell Lines Utilized

The following cell lines were obtained from the American Type TissueCulture Laboratory and cultured according to their specifications: Humanpancreatic cancer cell lines ASPC-1 and HS-766T.

Results

The results of the experiments are shown in FIG. 32, where a bar graphindicates the activity of a particular analog as measured by the % LDHrelease stimulated by each analog for 24 hours, with higher values ofLDH released indicating greater anti-neoplastic activity (apoptosis).All analogs were tested at a concentration of 25 μM. From these assays,it can be seen that some analogs, especially TOP-008 and TOP-009, showsignificantly greater activity than NCO-700 itself.

EXPERIMENT 4 Comparison of NCO-700 and Calpain Inhibitor in Cancer Cell

Death Assay

Based on Experiment 1 above, the anti-cancer activity of NCO-700 andCalpain inhibitor 1 (N-acetyl-leu-leu-norleucinal, C₂₀ H₃₇ N₃ O₄) wastested in cancer cell death assays of human cancer cell line DU-145.FIG. 33 shows data with calpain inhibitor 1 obtained fromBoehringer-Ingelheim, and NCO-700. In FIG. 33, NCO-700 is clearlysuperior to the other inhibitor. At a concentration of 100 μM, NCO-700is almost 10-fold more potent.

When an attempt of testing Calpain Inhibitor 1 in long-term assays (cellsurvival assay) was made, it was toxic to the cells, as would beexpected from its chemical structure, and the toxicity of CalpainInhibitor 1 would interfere with testing this compound in cell survivalassays or in vivo.

EXPERIMENT 5 Anti-neoplastic Activity of NCO-700, TOP-008, and RelatedAnalogs Alone ##STR31##

Based on Experiment 1 above, the anti-cancer activity of NCO-700,TOP-008, and analogs, TOP-201, 202, 203, 204, 205, 206, 207, and 007,was tested in cancer cell death assays of human cancer cell lineHS-578T. In FIGS. 34 (1,000 cells/well) and 35 (1,500 cells/well), a bargraph indicates the activity of a particular analog as measured by the %LDH released (DMSO, dimethyl sulfoxide=control), with higher values ofLDH released indicating greater anti-neoplastic activity. All analogswere tested at a concentration of 25 μM. From these assays it can beseen that TOP-008 (six month old (OLD) and newly synthesized (NEW)) hadvery good anti-neoplastic activity, and TOP-206 and NCO-700 had fairlygood anti-neoplastic activity. The other analogs tested also showed someanti-cancer activity. Sulfate forms appear to exhibit better results.

EXPERIMENT 6 Anti-Neoplastic Activity of TOP-008 in Apoptosis DetectionAssay

Anti-neoplastic activity of TOP-008 was tested using a commerciallyavailable kit, Apoptosis Detection Kit, from R & D SYSTEMS (Minnesota)in which annexin V, a member of the calcum and phospholipid bindingproteins, is used to detect apoptosis, following the protocolrecommended by the manufacturer. TOP-008 was tested at a concentrationof 50 μM, using Human breast cancer cell line HS-578T. Cells were washedin cold PBS twice and resuspended in a small volume of 1× bindingbuffer. Fluorescein-labeled annexin V and propidium iodide were added tothe cells. The cells expressing phosphatidylserine on the outer leafletof cell membranes bind annexin V and cells with a compromised cellmembrane allow propidium iodide to bind to the cellular DNA. Theresulting cells when immediately analyzed by flow cytometry can presentthree potential populations of cells: live cells which will not stainwith either fluorochrome, necrotic cells which will stain with bothfluorochrome, and cells undergoing apoptosis which will stain only withthe annexin V-FITC reagent. Analysis was performed on cytometersequipped with a single laser emitting excitation light at 488 nm. Theannexin V-FITC-generated signal was detected in the FITC signal detector(FL1). The results are shown in FIG. 36 in which the vertical axis isthe intensity of fluorescein emission, and the horizontal axis is thenumber of cells. FIG. 36a, 36b, and 36c show normal cancer cells(control), cancer cells with TOP-008 (50 μM), and cancer cells withTOP-008 (25 μM), respectively. FIG. 36 clearly shows that TOP-008induced apoptosis in human breast cancer cells.

EXPERIMENT 7 Apoptosis Induced DNA Fragmentation

PC-3, a prostate tumor cell line, was treated with 25 μM TOP-008, 50 μMNCO-700, or water for 48 hours. The DNA from each sample was purified byphenol extraction, and the fragmentation was resolved on an agarose gel.As a result, both samples treated with TOP-008 and NCO-700 showedsignificant DNA fragmentation while the control (treated with water) didnot. This fragmentation has been shown to be an indication of apoptosis(Jarvis et al., Canc. Res. 1154:1707-1714, 1994, Pandey et al., Biochem.Cell Biol. 72:625-629, 1994).

The foregoing experiments have established the new aspect of thepiperazine derivatives of the present invention, apoptosis, i.e., thederivatives are highly effective alone as an anti-neoplastic agent. Thefollowing experiment is an example which shows that the piperazinederivatives have significant anti-neoplastic activities when used inconjunction with standard chemotherapeutic agents such as vinblastineand adriamycin on drug-resistant tumors, even when the compounds are notsignificantly effective in apoptosis of certain cancer lines.

EXPERIMENT 8 Multidrug Resistance-Reversing Effect of NCO-700 in HumanNeoplasms

Methods and Results

In this Experiment, four different assays were utilized to measure theability of NCO-700 to block or reverse the drug-resistant phenotype ofcancer cells or tumors. These assays include:

1. Drug Accumulation Assays, where the effect of NCO-700 on the uptakeof radioactive (³ H)-vinblastine, an anticancer drug, is measured incultured cancer cells, that are both drug-resistant and drug-sensitive(Reference 3).

2. Cell Survival Assays, where the effect of NCO-700 to enhance cancercell killing by vinblastine was measured.

3. In Vivo Model of Tumorigenicity, where the effect of NCO-700 in theability of adriamycin to reduce tumor mass in drug-resistant neoplasmswas measured.

4. Human Tumor Biopsy Assays, where the effect of NCO-700 to enhancekilling, by either vinblastine or adriamycin, of cancer cells cultureddirectly from patient's tumors (Von Hoff et al., Cancer Res.43:1926-1931, 1983).

Cell Lines Utilized

In the first two in vitro assays, the drug accumulation assay and thecell survival assay, a human pharyngeal carcinoma cell line (KB-V-1)which is highly resistant to anticancer drugs was used. This cell linewas developed by Drs. Ira Pastan and Michael Gottesman of the NationalCancer Institute, by step-wise selection in colchicine of drug-resistantcells. The control cancer cell line (KB-3), from which the resistantcell lines were derived, is sensitive to anticancer drugs. The resistantKB-V-1 cell line is approximately 275 times more resistant tovinblastine than the sensitive KB-3 line and has a greatly amplified mdrgene which generates the observed resistance.

For the in vivo model of tumorigenicity, cell lines developed by Pastanand Gottesman which can grow as solid tumors in nude mice were utilized.This cell line was designated KB-CH 8-5, and was a nonrevertingresistant cell line. All of these cancer cell lines were generouslyprovided by Dr. Gottesman.

For the tumor biopsy assay, primary cell cultures were propagateddirectly from tumor specimens removed from patients. The cells weregrown in suspension culture, treated with adriamycin or vinblastine plusNCO-700, and plated onto soft agar and incubated for two weeks beforecell survival was determined.

Table 2 below summarizes results obtained in the drug accumulationassay. Measurement of ³ H!-vinblastine accumulation was performed by themethods developed in the laboratory of Drs. Gottesman and Pastan (Fojoet al., Cancer Res. 45:3002-3007, 1985). In this method, KB-V-1 and KB-3cells were plated in 10% fetal bovine serum at a density of 3×10⁵ cellsper well in 24-well Costar plates. The next day, the growth medium wasreplaced with Dulbecco's modified Eagle's medium with or without(control cells) 20 μM NCO-700. After ten minutes, the medium was removedand replaced with assay medium (0.5 ml) containing 0.1 μCi, (13 pmol) ³H!-vinblastine in the presence or absence of 20 μM NCO-700. After anadditional 30 min, the medium was removed, the plates were washed threetimes in ice-cold phosphate-buffered saline, the cells were detachedwith trypsin, and radioactivity determined by scintillation counting.

                  TABLE 2                                                         ______________________________________                                        NCO-700 STIMULATES THE ACCUMULATION OF                                        VINBLASTINE IN RESISTANT HUMAN CANCER CELLS                                   DRUG ACCUMULATION (pmole vinblastine/mg protein)                                            KB-V-1   KB-3                                                                 (resistant cells)                                                                      (sensitive cells)                                      ______________________________________                                        CONTROL (vinblastine                                                                          0.29 ± 0.03                                                                           2.99 ± 0.12                                     alone)                                                                        20 μM NCO-700 +                                                                            1.56 ± 0.03                                                                           3.17 ± 0.09                                     vinblastine                                                                   ______________________________________                                    

As shown in Table 2 above, the accumulation of the anticancer drug,vinblastine, was severely restricted in the drug-resistant cell line,KB-1, due to the action of the mdr pump. However, when this cell linewas exposed to 20 μM NCO-700, there was a 5-fold increase in the amountof vinblastine that remained in the cancer cell. As shown in Table 3below, this leads to an enhanced killing of the drug-resistant cells.

                  TABLE 3                                                         ______________________________________                                        NCO-700 INCREASES THE KILLING OF                                              RESISTANT CANCER CELLS BY VINBLASTINE                                                      VINBLASTINE (ng) to kill 50% of                                  CELL LINE    cells                                                            ______________________________________                                        KB-3 (sensitive)                                                                           3.2                                                              + 25 μM NCO-700                                                                         1.6                                                              KB-V-1 (resistant)                                                                         792                                                              + 25 μM NCO-700                                                                         148                                                              ______________________________________                                    

The cell survival experiments shown above were performed by plating KB-3(sensitive) and KB-V-1 (resistant) cells in 32 mm well plastic dishes ata cell density of 300-500 cells per well. Vinblastine and NCO-700 wereadded 16 hours after the initial cell plating. After 10 days ofincubation at 37°, cell colonies were stained and counted.

As shown in Table 3, there was a dramatic effect of NCO-700 on thesurvival of KB-V-1 resistant cancer cells. The levels of vinblastineneeded to kill 50% of the tumor cells dropped from 825 ng to 160 ng ofvinblastine when the resistant cells were co-incubated with 30 μMNCO-700. This effect reflects inhibition or blocking of the mdr pump,resulting in increased levels of anticancer drug in the cell.Interestingly, in this Experiment, there was also a slight enhancementof the killing of sensitive KB-3 cells as well.

In the next series of experiments, an in vivo model of tumorigenicitywas utilized to examine the effect of NCO-700 on tumor mass. Theseexperiments were performed with the cell line KB-CH-8/5, which is a cellline developed by Drs. Gottesman and Pastan, specifically for use inanimals as the cells from solid tumors in vivo. Nude male mice, 5-6weeks old and weighing between 25-30 g, were injected subcutaneouslywith 2-5×10⁶ KB-CH-8/5 cells and treated for 14 days with 0.6 mg/kgadriamycin and with or without NCO-700 in doses ranging from 0-80 mg/kgNCO-700 per day. The data shown in FIG. 37 were obtained by weighing thewell-encapsulated, excised tumor and recording the body weight of themice. Clearly shown in FIG. 37, the addition of NCO-700 significantlypotentiated the effect of adriamycin, especially at an amount of 40mg/kg or more.

In a final series of experiments, the effect of NCO-700 on survival ofcells cultured directly from biopsied patient tumors was performed. Thistechnique, referred to as a human tumor cloning system, was developed byVon Hoff and associates (Von Hoff et al., Cancer Res. 43:1926-1931,1983) and was used clinically to predict the course of chemotherapy forthese patients. In this method, tumor biopsies were separated into cellsuspensions and treated for 1 hour with either 0.4 mg/ml vinblastine(VLB) or 0.5 mg/ml adriamycin (ADR) plus or minus 20 μM NCO-700. Thecell suspensions were washed after one hour of incubation and platedwith soft agar. After 14 days of incubation at 37°, the percent ofcolonies surviving was scored. As a result, NCO-700 had significantactivity in inhibiting the survival of certain human tumor cells. In twotumors tested from patients with breast cancer who were receivingchemotherapy, NCO-700 significantly sensitized these tumor cells to theactions of adriamycin (ADR) and vinblastine (VLB). In particular, thesurvival of tumor cells decreased to approximately 1/2 to 1/3 afterexposure to NCO-700 and adriamycin. All other tumors tested showedvarying degrees of positive response. Excellent responses were seen witha kidney tumor treated with NCO-700 and adriamycin, where the survivalof the tumor cells decreased to approximately 1/4 and with an ovariantumor treated with NCO-700 and vinblastine where the survival decreasedto approximately 1/3. The prediction on the mechanism of how NCO-700 issensitizing these tumor cells to the anticancer drugs would be thatNCO-700 is blocking or inhibiting the mdr pump from effluxing the cancerdrug from the cell.

Conclusion

NCO-700 stimulated by 5-fold the accumulation of the anticancer drug,vinblastine, in human carcinoma cell lines. This resulted in a highlysignificant increase in the killing of these cancer cells. When NCO-700was administered with adriamycin to nude mice carrying resistant humantumors (KB-CH 8-5), there was a decline, by 60%, of tumor mass withoutany corresponding loss of body weight, although NCO-700 was notsignificantly effective on its own. Finally, NCO-700 showed excellentactivity in sensitizing to anticancer drugs, a number of primary tumorcell lines grown from patient biopsies.

The above experiment is an example which shows that the piperazinederivatives have significant anti-neoplastic activities when used inconjunction with standard chemotherapeutic agents such as vinblastineand adriamycin on drug-resistant tumors, even when the compounds are notsignificantly effective in apoptosis of certain cancer lines. Thus, as aclinical trial, first, a compound of the present piperazine derivativesis administered as a primary chemotherapeutic agent to a patient havingcancer (irrespective of the existence of multidrug resistance), and whenthe compound does not appear to be effective on its own, anotherchemotherapy is additionally conducted as adjunctive therapy, e.g., theadministration of other chemotherapeutic agents substantiallycontemporaneously with the present piperazine derivatives.

It will be understood by those of skill in the art that numerousvariations and modifications can be made without departing from thespirit of the present invention. Therefore, it should be clearlyunderstood that the forms of the present invention are illustrative onlyand are not intended to limit the scope of the present invention.

What is claimed is:
 1. A method for inducing cell death in neoplasticcells, comprising:administering a compound of formula I or apharmaceutically acceptable salt thereof to a patient having neoplasticcells sensitive to said compound or said salt, in an amount sufficientto induce cell death in said neoplastic cells: ##STR32## where R¹ ishydroxyl or C1-4 alkoxyl, R² is C3-4 alkyl, R³ is C1-4 alkyl, ##STR33##in which n is an integer of 0 to
 3. 2. The method according to claim 1,wherein said compound is of the following formula: ##STR34## where R⁴ isC₂ H₅ O--.
 3. The method according to claim 1, wherein said compound isof the following formula: ##STR35## where R⁴ is C₂ H₅ O--.
 4. The methodaccording to claim 1, wherein said compound is in the form of sulfate.5. The method according to claim 1, wherein said neoplastic cells to betreated are selected from the group consisting of human breast cancercells, human melanoma cells, human ovarian cancer cells, human coloncancer cells, human pancreatic cancer cells, and human prostate cancercells.
 6. The method according to claim 1, wherein said neoplastic cellsto be treated are undifferentiated cancer cells.
 7. The method accordingto claim 1, wherein said neoplastic cells to be treated carry an activemdr gene.
 8. A method for treating neoplastic cells, consistingessentially of:administering a compound of formula I or apharmaceutically acceptable salt thereof to a patient having neoplasticcells sensitive to said compound or said salt, in an amount sufficientto induce cell death in said neoplastic cells: ##STR36## where R¹ ishydroxyl or C1-4 alkoxyl, R² is C3-4 alkyl, R³ is C1-4 alkyl, ##STR37##in which n is an integer of 0 to
 3. 9. The method according to claim 8,wherein said compound is of the following formula: ##STR38## where R⁴ isC₂ H₅ O--.
 10. The method according to claim 8, wherein said compound isof the following formula: ##STR39## where R⁴ is C₂ H₅ O--.
 11. Themethod according to claim 8, wherein said compound is in the form ofsulfate.
 12. The method according to claim 8, wherein said neoplasticcells to be treated are selected from the group consisting of humanbreast cancer cells, human melanoma cells, human ovarian cancer cells,human colon cancer cells, human pancreatic cancer cells, and humanprostate cancer cells.
 13. The method according to claim 8, wherein saidneoplastic cells to be treated are undifferentiated cancer cells. 14.The method according to claim 8, wherein said neoplastic cells to betreated carry an active mdr gene.